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An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.

Ghosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR - J. Cell Biol. (1998)

Bottom Line: When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min.Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes.TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.

ABSTRACT
To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

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Endocytosed fluorescent anti-Tac antibodies traffic to  the TGN. Cells were labeled with Cy3 conjugated anti-Tac Fab  for 10 min at 37°C and then chased for 40 min. The cells were  fixed and labeled with C6-NBD-ceramide to identify the TGN.  (a) A single confocal microscope section showing the C6NBD-ceramide staining; (b) Cy3-Fab labeling. These images show that  the endocytosed Fab reaches the TGN (arrows). (c and d) Images  taken by wide-field microscopy of C6-NBD-ceramide (c) and  Cy3-IgG (d) show that endocytosed Cy3-IgG can also reach the  TGN (arrows). (e and f) Cells were incubated with F-Tf at 37°C  for 30 min to label the ERC (green), and then they were fixed,  permeabilized, and labeled with Cy3-IgG to identify the TGN  (red). A 3-D stack of images through the cell was obtained by  confocal microscopy. (f) An x-y maximum projection of this  stack, and (e) a y-z maximum projection of the same cells, which  is similar to viewing the cells from the right-hand side. The arrow  indicates that although a part of the TGN may appear colocalized  with the ERC in an x-y projection (f), the y-z projection shows  that it is in a different vertical position (e). In these cells the TGN  is to the side and above the ERC. Bar, 10 μm.
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Figure 1: Endocytosed fluorescent anti-Tac antibodies traffic to the TGN. Cells were labeled with Cy3 conjugated anti-Tac Fab for 10 min at 37°C and then chased for 40 min. The cells were fixed and labeled with C6-NBD-ceramide to identify the TGN. (a) A single confocal microscope section showing the C6NBD-ceramide staining; (b) Cy3-Fab labeling. These images show that the endocytosed Fab reaches the TGN (arrows). (c and d) Images taken by wide-field microscopy of C6-NBD-ceramide (c) and Cy3-IgG (d) show that endocytosed Cy3-IgG can also reach the TGN (arrows). (e and f) Cells were incubated with F-Tf at 37°C for 30 min to label the ERC (green), and then they were fixed, permeabilized, and labeled with Cy3-IgG to identify the TGN (red). A 3-D stack of images through the cell was obtained by confocal microscopy. (f) An x-y maximum projection of this stack, and (e) a y-z maximum projection of the same cells, which is similar to viewing the cells from the right-hand side. The arrow indicates that although a part of the TGN may appear colocalized with the ERC in an x-y projection (f), the y-z projection shows that it is in a different vertical position (e). In these cells the TGN is to the side and above the ERC. Bar, 10 μm.

Mentions: TRVb1 cells were transfected with TacTGN38, and clonal cell lines were selected by FACS sorting using Cy3-labeled monoclonal anti-Tac IgG. We first examined the distribution of internalized Cy3-labeled anti-Tac IgG and monovalent Fab. Fig. 1 shows confocal microscopy images of Cy3-Fab (Fig. 1 b) and Cy3-IgG (Fig. 1 d) after a 5–10 min incubation with the antibodies and a 30–40-min chase. The cells were also stained with C6-NBD-ceramide which labels the TGN (36), and single optical sections were obtained in regions that showed significant NBD fluorescence (Fig. 1, a and b). Both the IgG and the Fab showed extensive colocalization with the C6-NBD-ceramide, demonstrating that the endocytosed Cy3 labeled anti-Tac Fab and IgG reached the TGN. After an extensive chase, internalized anti-Tac also codistributed with furin, an endoprotease that is localized to the TGN (see Fig. 4, c and d). Endocytosed TacTGN38 trafficked to the TGN in all the clonal lines we examined.


An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.

Ghosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR - J. Cell Biol. (1998)

Endocytosed fluorescent anti-Tac antibodies traffic to  the TGN. Cells were labeled with Cy3 conjugated anti-Tac Fab  for 10 min at 37°C and then chased for 40 min. The cells were  fixed and labeled with C6-NBD-ceramide to identify the TGN.  (a) A single confocal microscope section showing the C6NBD-ceramide staining; (b) Cy3-Fab labeling. These images show that  the endocytosed Fab reaches the TGN (arrows). (c and d) Images  taken by wide-field microscopy of C6-NBD-ceramide (c) and  Cy3-IgG (d) show that endocytosed Cy3-IgG can also reach the  TGN (arrows). (e and f) Cells were incubated with F-Tf at 37°C  for 30 min to label the ERC (green), and then they were fixed,  permeabilized, and labeled with Cy3-IgG to identify the TGN  (red). A 3-D stack of images through the cell was obtained by  confocal microscopy. (f) An x-y maximum projection of this  stack, and (e) a y-z maximum projection of the same cells, which  is similar to viewing the cells from the right-hand side. The arrow  indicates that although a part of the TGN may appear colocalized  with the ERC in an x-y projection (f), the y-z projection shows  that it is in a different vertical position (e). In these cells the TGN  is to the side and above the ERC. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 1: Endocytosed fluorescent anti-Tac antibodies traffic to the TGN. Cells were labeled with Cy3 conjugated anti-Tac Fab for 10 min at 37°C and then chased for 40 min. The cells were fixed and labeled with C6-NBD-ceramide to identify the TGN. (a) A single confocal microscope section showing the C6NBD-ceramide staining; (b) Cy3-Fab labeling. These images show that the endocytosed Fab reaches the TGN (arrows). (c and d) Images taken by wide-field microscopy of C6-NBD-ceramide (c) and Cy3-IgG (d) show that endocytosed Cy3-IgG can also reach the TGN (arrows). (e and f) Cells were incubated with F-Tf at 37°C for 30 min to label the ERC (green), and then they were fixed, permeabilized, and labeled with Cy3-IgG to identify the TGN (red). A 3-D stack of images through the cell was obtained by confocal microscopy. (f) An x-y maximum projection of this stack, and (e) a y-z maximum projection of the same cells, which is similar to viewing the cells from the right-hand side. The arrow indicates that although a part of the TGN may appear colocalized with the ERC in an x-y projection (f), the y-z projection shows that it is in a different vertical position (e). In these cells the TGN is to the side and above the ERC. Bar, 10 μm.
Mentions: TRVb1 cells were transfected with TacTGN38, and clonal cell lines were selected by FACS sorting using Cy3-labeled monoclonal anti-Tac IgG. We first examined the distribution of internalized Cy3-labeled anti-Tac IgG and monovalent Fab. Fig. 1 shows confocal microscopy images of Cy3-Fab (Fig. 1 b) and Cy3-IgG (Fig. 1 d) after a 5–10 min incubation with the antibodies and a 30–40-min chase. The cells were also stained with C6-NBD-ceramide which labels the TGN (36), and single optical sections were obtained in regions that showed significant NBD fluorescence (Fig. 1, a and b). Both the IgG and the Fab showed extensive colocalization with the C6-NBD-ceramide, demonstrating that the endocytosed Cy3 labeled anti-Tac Fab and IgG reached the TGN. After an extensive chase, internalized anti-Tac also codistributed with furin, an endoprotease that is localized to the TGN (see Fig. 4, c and d). Endocytosed TacTGN38 trafficked to the TGN in all the clonal lines we examined.

Bottom Line: When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min.Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes.TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.

ABSTRACT
To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

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